Gastrointestinal stromal tumor (GIST) treated with targeted therapies such as imatinib mesylate (IM), sunitinib, and regorafenib, generally escape disease control and progress over time. The current standard of care for advanced inoperable GIST involves the sequential application of these inhibitors that target the activated forms of the KIT or PDGFRA receptors found in GIST. We hypothesized that inhibiting additional molecular targets in combination with IM may provide more substantial disease control. Recent studies have implicated the PI3- kinase/AKT pathway in survival of IM-resistant GIST cell lines and tumors. We therefore challenged IM- sensitive GIST xenografts with a novel combination of IM and an AKT inhibitor to test if the combination enhanced disease stabilization afforded by front-line IM therapy. In these preliminary studies, the combination therapy provided significantly improved efficacy as compared to treatment with monotherapy alone, as measured by tumor response and animal survival. These results provide justification for additional pre-clinical studies challenging various GIST xenografts modeling both intrinsic and acquired IM resistance, as described in this proposal. To explore the molecular aspects of tumor response to this novel combination, we carried out RNAseq studies on these xenografts, in the hope of identifying additional therapeutic targets for GIST. This analysis was fruitful, identifying two genes, brain expressed, X-linked 1 (BEX1) and neuronal pentraxin I (NPTX1), whose expression was significantly up-regulated only in combination-treated tumors. BEX1 and NPTX1 have been implicated as tumor-suppressor genes in various cancers, and with the induction of apoptotic pathways in diverse cell types. Specifically, BEX1 acts as a BCL-2 antagonist, inducing apoptosis by binding BCL-2 and suppressing the formation of anti-apoptotic BCL-2/BAX heterodimers, while NPTX1 expression has been linked to enhanced mitochondrial translocation of pro-apoptotic BAD and BAX proteins and enhanced neuronal cell death. We hypothesize that this combination results in induction of these two genes that influence tumor cell fate by shifting the pro/anti-apoptotic balance towards cell death via the BCL-2, BAD/BAX pathways, and may provide additional avenues to approach GIST treatment. This proposal seeks to functionally test this central hypothesis, as well as to evaluate targeting members of these pathways in both IM- sensitive and ?resistant GIST xenograft models. The proposed studies are designed to provide support for potential application of these novel therapeutic modalities in the clinical setting.